Chemische Charakterisierung und Spurenanalytik
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Introduction to microplastics, definition, relevance, analytical approaches & challenges. TED-GC/MS working principle, features & performance. Optimisation in TED-GC/MS methodology, new validation data. Replacement of Internal Standard, outlook towards upcoming publication and application within EU´s revised drinking water directive.
Accreditation of analytical methods, either according to GxP or ISO regulations, requires a comprehensive quality management system. General quality documents are often already in place, which need to be extended by method-specific documentation. In this presentation we like to show an idea of a modular set of standard operating procedures (SOP) specifically developed meeting the requirements for quantitative NMR. The future goal is to collaborate with different accreditated NMR laboratories to compile a universal set of SOP and other quality documents that can be modified and used as a starting point for developing your own quality system for applications of qNMR in a regulated environment.
Nano-carrier systems such as liposomes have promising biomedical applications. Nevertheless, characterization of these complex samples is a challenging analytical task. In this study a coupled hydrodynamic chromatography-single particle-inductively coupled plasma mass spectrometry (HDC-spICP-MS) approach was validated based on the technical specification (TS) 19590:2017 of the international organization for standardization (ISO). The TS has been adapted to the hyphenated setup. The quality criteria (QC), e.g., linearity of the calibration, transport efficiency, were investigated. Furthermore, a cross calibration of the particle size was performed with values from dynamic light scattering (DLS) and transmission electron microscopy (TEM). Due to an additional Y-piece, an online-calibration routine was implemented. This approach allows the calibration of the ICP-MS during the dead time of the chromatography run, to reduce the required time and enhance the robustness of the results. The optimized method was tested with different gold nanoparticle (Au-NP) mixtures to investigate the characterization properties of HDC separations for samples with increasing complexity. Additionally, the technique was successfully applied to simultaneously determine both the hydrodynamic radius and the Au-NP content in liposomes. With the established hyphenated setup, it was possible to distinguish between different subpopulations with various NP loads and different hydrodynamic diameters inside the liposome carriers.
Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the sticky character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing an LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend an LC-MS/MS-based quantification method with a dynamic range of 0.5–40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials.
Hepcidin-25 has attracted much attention ever since its discovery in 2001. It is widely recognized that this peptide hormone plays a major role in the regulation of iron levels in mammals and can reveal important clinical information about several iron-related disorders. However, the development of a reliable assay to quantify hepcidin proved to be problematic and serum hepcidin-25 concentrations determined by various assays differ substantially. Challenges arise in the MS analysis of hepcidin due to the “sticky” character of the peptide and the lack of suitable standards.
With the aim to tackle the current difficulties in hepcidin quantification and improve the status of this promising biomarker in the clinical field, we developed a rapid and robust analytical strategy for the quantification of hepcidin-25 in human samples based on HPLC-MS/MS (QqQ) as a reference method candidate to be implemented in routine laboratories. The novelty of the method is the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces. Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions where the use of acidic mobile phases was compared with a novel approach involving solvents at high pH containing 0.1% of ammonia. Both methods were carefully validated and applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with very good correlation of the results.